Plasma / Serum Protein Binding Determinations
Michael J. Banker and Tracey H. Clark  Current Drug Metabolism, 2008, 9, 854-859

Abstract

The binding of a drug to serum or plasma proteins enables the transport of drugs via the blood to sites of action throughout the
body. For expediency we will use serum proteins throughout this discussion with the understanding that one can substitute the term
plasma proteins in all experimental instances. Only the fraction of drug unbound from serum proteins is available to diffuse from the vascular
system and accumulate in tissues thereby enabling interaction with therapeutic targets and accessibility to xenobiotic clearance
pathways. Therefore, the extent of drug binding to serum proteins can have a significant impact on pharmacokinetic (PK) parameters
such as clearance rates and volume of distribution. In addition, because only the unbound drug is available to interact with therapeutic
targets, the extent of serum binding can have significant effects on the pharmacodynamic properties of a compound as well [1, 2] Determining
the fraction of drug bound to serum proteins is a standard parameter evaluated in the process of drug discovery. Although the
clinical importance of changes in serum protein binding has been questioned [3-8] the need for serum protein binding studies in the discovery
and preclinical development stages is essential for the pharmacokinetic modeling of drugs[1, 3, 9]. The extent of serum protein
binding is an important parameter used in many in vivo modeling calculations to estimate the volume of distribution, organ clearance, and
for scale-up of pharmacokinetic and pharmacodynamic parameters from animal models to humans [3, 10, 11]. The convergence of several
trends in the pharmaceutical industry including high speed chemical synthesis technologies, the increasing use of in silico ADME
modeling together with early in vivo evaluations of lead compounds has increased the demand for serum protein binding determinations[
12].

 DEVELOPMENT AND VALIDATION OF A 96-WELL EQUILIBRIUM DIALYSIS APPARATUS FOR MEASURING PLASMA PROTEIN BINDING
Michael J. Banker, Tracey H. Clark, John A. Williams J Pharm Sci 2003 92:967-974.

  
Abstract

A 96-well equilibrium dialysis block was designed and constructed that is compatible with most standard 96-well format laboratory supplies and instruments.  The unique design of the dialysis apparatus allows one to dispense and aspirate from either or both the sample and dialysate sides from the top of the apparatus, which is not possible with systems currently on the market.  This permits the investigator to analyze a large number of samples, time points, or replicates in the same experiment.  The novel alignment of the dialysis membrane vertically in the well maximizes the surface to volume ratio, eliminates problems associated with trapped air pockets and allows one to add or remove samples independently or all at once.  Furthermore, the design of the apparatus allows both the sample and dialysate sides of the dialysis well to be accessible by robotic systems so assays can be readily automated.  Teflon construction is used to minimize non-specific binding of test samples to the apparatus.  The device is reusable, easily assembled and can be shaken in controlled temperature environments to decrease the time required to reach equilibrium as well as facilitating dissolution of test compounds.  Plasma protein binding values obtained for ten diverse compounds using standard dialysis equipment and the 96-well dialysis block validates this method.

IMPACT OF NONSPECIFIC BINDING TO MICROSOMES AND PHOSPHOLIPIDS ON THE INHIBITION OF CYTOCHROME P4502D6: IMPLICATIONS FOR RELATING IN VITRO INHIBITION DATA TO IN VIVO DRUG INTERACTIONS
Jeannine M. Margolis and R. Scott Obach   Drug Metab Dispos 2003 31: 606-611.

Abstract

The effects of microsomal concentration on the inhibitory potencies of four compounds fluoxetine, quinidine, imipramine, and ezlopitant on heterologously expressed recombinant CYP2D6-catalyzed bufuralol 1'-hydroxylase activity were determined. Increasing microsomal concentration from 0.0088 to 2.0 mg/ml, using additional microsomes not containing cytochrome P450, resulted in a marked increase in IC50 and KI values for fluoxetine, ezlopitant, and imipramine, when inhibition constants were calculated using the nominal concentration of inhibitor added to the incubation mixture. The extent of nonspecific binding of these inhibitors to microsomes was determined using equilibrium dialysis. The extent of binding increased with increasing microsomal concentration. Binding was greatest for ezlopitant, followed by fluoxetine, imipramine, and quinidine. Correcting inhibition constants for the extent of nonspecific binding resulted in greater consistency of these values with differing microsomal protein concentrations. This effect was also studied with added phospholipid. Inhibition constants increased with increasing phospholipid, and nonspecific binding was also observed for these four drugs to phospholipid. This suggests that the phospholipid component of microsomes possesses some or all of the responsibility for nonspecific binding, and its effect on inhibitors of drug-metabolizing enzymes. These findings suggest that inhibition constants for drugs as inhibitors of microsomal drug-metabolizing enzymes, such as cytochrome P450, should be corrected for the extent of nonspecific binding to components of the in vitro matrix. The implications of this on the prediction of drug-drug interactions from in vitro data are discussed.

In Silico Modeling of Non-specific Binding to Human Liver Microsomes
Hua Gao, Lili Yao, Heather W. Mathieu, Ying Zhang, Tristan S. Maurer,
Matthew D. Troutman, Dennis O. Scott, Roger B. Ruggeri and Jing Lin

Impact of Physicochemical and Structural Properties on the Pharmacokinetics of a Series of  1L-Adrenoceptor Antagonists
Alison Betts, Fidelma Atkinson, Iain Gardner, David Fox, Rob Webster, Kevin Beaumont, and Paul Morgan
(Received February 14, 2007; accepted May 11, 2007)

Current Protocols in Pharmacology  UNIT 7.5   Determination of Compound Binding to Plasma Proteins
Natasha Dow (Covance Laboratories, Inc., Madison, Wisconsin)
Print Publication Date: September, 2006

Semi-Automated Plasma Protein Binding Assay Utilizing a 96-well Equilibrium Dialyzer and a Mixed Matrix Approach Tracks: Contributed Papers: 102 Other Pharmacokinetics and Pharmacodynamics
Emile G. Plise, Dr. Laurent Salphati, and Daniel Tran Genentech

Abstract

The determination of plasma protein binding to new chemical entities is most frequently determined by the equilibrium dialysis method. The typical apparatus is large, requires large volumes of both plasma and buffer, and limited to 20 samples per apparatus at a time. We have validated a 96-well protein binding apparatus (HTDialysis, LLC) that provides the ability to run more samples in the same time frame using less volume. Studies were undertaken to compare the two methods in different species using several well-known fluorescent probes (2-hydroxy 4-trifluoro-coumarin, resorufin, fluorescein) and common drugs. The results demonstrate that the two methods yield comparable data. The new 96-well method requires less preparation time and reagents and costs less, while providing data that are equivalent and reproducible as those obtained with the older method. Additionally, the ability to use fluorescent probes as positive controls for plasma protein binding studies also provides an inexpensive and convenient method of confirming the validity of a particular plasma protein binding experiment.
 Introduction
A study was undertaken to validate a new time and cost-efficient 96-well protein binding apparatus. Five commercially available drugs with a wide range of reported percent protein binding values and encompassing a diverse range of therapeutic indications were selected. The equivalence of the new high-throughput method to the old single-cell method was first established

Azithromycin/Chloroquine Combination Does Not Increase Cardiac Instability despite an Increase in Monophasic Action Potential Duration in the Anesthetized Guinea Pig
Anthony A. Fossa, Todd Wisialowski, J. Neil Duncan, Shibing Deng, and Michael Dunne
Am. J. Trop. Med. Hyg., 77(5), 2007, pp. 929-938 Copyright © 2007 by The American Society of Tropical Medicine and Hygiene

Studies of drug binding to plasma proteins using a variant of equilibrium dialysis
Mats A.L. Eriksson, Johan Gabrielsson, and Lars B. Nilsson
Journal of Pharmaceutical and Biomedical Analysis Volume 38, Issue 3, 1 July 2005, Pages 381-389 

The Important Role of Bcrp (Abcg2) in the Biliary Excretion of Sulfate and Glucuronide Metabolites of Acetaminophen, 4-Methylumbelliferone, and Harmol in Mice
Maciej J. Zamek-Gliszczynski, Ken-ichi Nezasa, Xianbin Tian, J. Cory Kalvass, Nita J. Patel, Thomas J. Raub, and Kim L. R. Brouwer
Mol Pharmacol 70:2127-2133, 2006